The primary goal of the proposed investigation is the detailed elucidation[unreadable] of the molecular aspects of toxin regulation in V. cholerae. Biochemical[unreadable] and genetic methods will be used to locate the DNA sites involved in[unreadable] formation of the cholera toxin operon (ctx) promoter and control sites. We[unreadable] will attempt to define the precise molecular mechanism by which the[unreadable] positive regulatory gene toxR, activates ctx transcription. The regulatory[unreadable] role played by the repetitive sequence TTTTGAT will be studied by localized[unreadable] and mismatch primer mutagenesis. The nutritional and physical factors[unreadable] which regulate toxin production will be correlated with specific[unreadable] transcriptional effects on ctx or toxR. We will continue to study ctx[unreadable] amplification as a genetic mechanism controlling toxin production in E1 Tor[unreadable] strains of V. cholerae. In this regard, we will test the repetitive[unreadable] sequence RS1 for its ability to mediate various recombinational events[unreadable] including transposition, duplication and site-specific recombination. The[unreadable] knowledge gained from all the above studies will be used to devise methods[unreadable] with which to study toxin regulation and ctx amplification in vivo.[unreadable] Specifically, we will study how the intestine provides a selective[unreadable] environment for increased toxin expression by defining which genes (ctxA,[unreadable] ctxB or toxR) are necessary for in vivo selection of genetic reversion and[unreadable] amplification events. We will apply what we have learned about ctx[unreadable] regulation and expression to the development of improved live and dead,[unreadable] oral vaccines against cholera. Mutations will be constructed in the cloned[unreadable] recA gene of V. cholerae and then recombined back in place of the rec+ gene[unreadable] of the prototype live oral vaccine strain 0395-N1. In addition to recA,[unreadable] streptomycin-dependent, DAP- and ga1E mutations will also be incorporated[unreadable] into 0395-N1 derivatives as a means of limiting the persistence of this[unreadable] strain in humans and the environment. Finally, derivatives of 0395-N1[unreadable] which are suitable for use as a dead, oral cholera vaccines will be[unreadable] constructed which hyperproduce the B subunit of the toxin in both a[unreadable] secreted and nonsecreted form.[unreadable]